Antibacterial activity of lactobacilli probiotics on clinical strains of Helicobacter pylori

Objective(s): Treatment of Helicobacter pylori infection by common drugs may be associated with several problems such as antimicrobial resistance to commonly used antibiotics and side effects of employed drugs. Therefore, exploration of non-chemical compounds which are safer than chemical ones is becoming important as an alternative therapy. The purpose of this study was to evaluate the effects of lactic acid bacteria (LAB) against clinical strains of H. pylori. Materials and Methods: Study of antibacterial effects of LAB against H. pylori strains included: evaluation of LAB effects as well as its cell-free supernatant (CFS) to reduce the number of H. pylori, and to examine the effects of CFS to inhibit the urease activity of H. pylori. The anti-adhesion effect of LAB on adherence of H. pylori to epithelial cell line was also evaluated. Results: Evaluation of the anti H. pylori effect of LAB depended on the strain of H. pylori and Lactobacillus. However, CFS of LAB reduced significantly the growth of all H. pylori strains. Also, urease activity of H. pylori strains was inhibited by CFS of LAB demonstrating that their organic acid may have a role in this inhibition. The significant anti-adhesion effect of LAB on adherence of H. pylori was also observed. Conclusion: Presence of LAB and/or their CFS can reduce the count of H. pylori, inhibit the urease activity of H. pylori, and reduce adhesion of H. pylori to epithelial cell line. This may be important for the impact of H. pylori colonization in the host stomach.


Introduction
Bacterial interference between the natural flora and the pathogens was proposed in the 20 th century and following this finding, it was investigated for the control of infections. The investigators have demonstrated that the probiotics such as lactobacilli can prevent the growth of a wide range of human and animal microbial species which are their pathogens. Of them; the effects of Lactic Acid Bacteria (LAB) on Helicobacter pylori may be noted (1,2). H. pylori has been known as a major cause of chronic gastritis, peptic ulcer disease and stomach cancer (3,4). Bacterial virulence factors help H. pylori to invade the host stomach, cause disease and evade the host defenses. Expression of several virulence factors such as urease, adhesion factors, vacuolating cytotoxin A, and the cytotoxin-associated gene A, are associated with pathogenicity of H. pylori (5). The standard triple therapy regimen for treatment of H. pylori infection was widely used throughout the world (6). This treatment consisted of a proton pump inhibitor (PPI) plus two antibiotics (clarithromycin and amoxicillin) (7). The success of this treatment regimen has become progressively decreased in recent years. Non-compliance and the emergence of antibiotic-resistant strains of H. pylori are considered as the major factors contributing to treatment failure (8). Regarding the side effects of antibiotics in majority of the cases and emergence of resistant bacteria in the stomach of the infected patients, use of living microbial agents, such as LAB (as probiotics bacteria), may be a useful alternative in preventing the symptoms of H. pylori infection via inhibiting its growth by competing with this pathogens (9). Probiotics are definde as live microorganisms, which when administered in enough amount give a health benefit on the host. Probiotics have diverse mechanisms for inhibition of pathogenic bacteria, e.g. nutrient competition, production of inhibitory compound (bacteriocins, organic acids, biosurfactant,…) , immunostimulation and competition for binding sites (10).
The purpose of present study was to investigate the inhibitory effects of six species of LAB and their CFS against growth, urease (in vitro) and adhesion to epithelial cell line of eight clinical strains of H. pylori which were selected according to their diverse antibiotic resistance profiles.

Materials and Methods
Bacterial strains and culture conditions: H. pylori strains used in this study were selected according to their susceptibility profile that were in H. pylori Lab at Alzahra University (Table 1). They were grown at 37 °C for 48 hr under microaerobic conditions by Gas pack C (Merck) on Brucella Agar containing 5-10 % defibrinated sheep blood supplemented with vancomycin (10 μg/ml), polymyxin (2.5 IU/ml) and amphotericin B (2 μg/ml). Antibiotic susceptibility testing of H. pylori strains was performed according to agar disk-diffusion procedure (11) in a microaerobic condition for 72 hr, then their inhibition zone diameters were examined. The zones of inhibitions were interpreted according to those of the previously described protocol (12)(13)(14)(15).
LAB corresponded to Lactobacillus acidophilus ATCC 4356, Lactobacillus rhamnosus ATCC 7469, Lactobacillus reuteri ATCC 23272, Lactobacillus fermentum ATCC 9338, Lactobacillus plantarum ATCC 8014 and Lactobacillus casei ATCC 39392 that were provided from Iranian Research Organization for Science and Technology (IROST). These LAB were selected since their antibacterial effects on the most photogenic bacteria has been confirmed in our previous researches (10). LAB were cultured in DeMan-Rogosa-Sharpe (MRS) broth for 48 hr at 37 °C . CFS of LAB were prepared from their 48 hr culture by centrifugation at 10000×g for 10 min at 4 °C , and by filtration through the 0.22-μmpore-size filter (Millipore).

Cell line
The gastric epithelial cell line AGS (ATCC CRL-1739) was cultured in RPMI 1640 (Dacell) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% PenStrep (100 U/ml penicillin and 100 µg/ml streptomycin, bioidea). The cells were maintained at 37 °C and 5% CO 2 in a humidified environment. The day before of experiment, the cells were seeded into tissue culture plates to form a monolayer overnight. At the start of each experiment, the cell culture medium was replaced with RPMI 1640 with 3% serum and without antibiotics.

Effect of lactobacilli CFS on H. pylori
Agar diffusion assay H. pylori suspensions equivalent to 2 McFarland, were plated on Mueller-Hinton agar plates containing 5 % defibrinated sheep blood without antibiotics. Wells (diameters, 6 mm) were drilled into the agar using the sterile Pasteur pipettes. They were filled with 100 μl CFS of LAB. Plates were incubated for 72 hr under microaerobic conditions at 37 °C , and the diameters of inhibition zones around the wells were measured according to the previously described protocol (16).
Liquid culture assay H. pylori suspension (10 8 CFU/ml) were prepared in brain heart broth without antibiotics and were incubated under microaerobic conditions at 37 °C with CFS (equal volume); MRS broth medium was used as the control. The viability of H. pylori after 48 hr was evaluated by determination of the viable count (CFU) on MHA agar containing 5% defibrinated sheep blood plates following incubation at 37 °C under microaerobic conditions (16).

Effect of lactobacilli CFS on urease activity of H. pylori strains The urease activity of H. pylori strains
Urease activity was determined in medium containing, phosphate buffer, urea and 0.012% phenol red (as indicator of pH). The principle of this analysis was the production of ammonia due to hydrolysis of urea, which was measured by absorbance of produced color at 550 nm by the spectrophotometer. Optimization of the reaction conditions concerning urea concentration, pH of reaction buffer and temperature for urease activity of H. pylori was performed before analysis of urease activity. For this purpose, the optimum effect of urea concentration (15 to 25 mg/ml), pH (5.8 to 7.8 pH), and temperature (37 to 55 °C ) was examined by response surface methodology (RSM) (17). In all cases, 50 ul of H. pylori cells, grown in the Brucella broth for 48 hr at 37 °C (initial number of bacteria: 10 8 CFU/ml) was added to 1 ml of urease reaction buffer and the color absorbance was measured at 550 nm after incubation for 1 hr (18)(19)(20).

The inhibitory effect of lactobacilli CFS on urease activity of H. pylori
For analysis of urease inhibitory effects by LAB, H. pylori cells incubated in Brucella broth at 37 °C (initial number: 10 8 CFU/ml) for 48 hr, were mixed with various concentrations of CFS (10 to 40%) and incubated at 37 °C for 30, 60, and 120 min. The resulted color was read at 550 nm by spectrophotometer according to the previously described protocol (16). MRS broth was used as control of reaction. Also, the effect of CFS that could be neutralized by NaOH (1 mol/lit) to 7.0±0.01 and acids such as acetic acid (Merck) and lactic acid (Merck) on urease activity were evaluated (21).

Effect of LAB on the count of H. pylori strains by coculture
H. pylori strains also LAB were recovered from plates and were suspended in BHB with 10 % (v/v) horse serum and MRS broth, respectively (McFarland No. 1 and 0.5 respectively). Equal volumes of each suspension was combined and incubated at 37 °C at atmosphere containing 5% CO 2 . After 48 hr incubation at 37 °C under microaerobic conditions, the number of CFU was evaluated on MHA agar containing 5% defibrinated sheep blood which was selective for the growth of H. pylori containing 5 µg/ml trimethoprim, 2.5 U/ml polymyxin B, 10 µg/ml vancomycin and 5 µg/ ml penicillin (Sigma-Aldrich) (the LAB were sensitive to penicillin but H. pylori was resistance) (22).

Effect of LAB on adhesion of H. pylori to AGS cell line
Bacterial suspensions were prepared from the culture plates of H. pylori strains and adjusted to McFarland tube No 3. After centrifugation, bacterial cells were suspended in RPMI 1640 with 3% serum. As well, bacterial suspensions were prepared from the overnight culture plates of LAB and adjusted to McFarland tube no 0.5; after centrifugation, bacterial cells were suspended in RPMI 1640 with 3% serum. Epithelial cells grown in 48-well plates were infected with both H. pylori strains and LAB at an multiplicity (MOI) of 100 for each species. After 3 hr of incubation, the cells were washed three times with phosphate-buffered saline (PBS) to remove any unbound bacteria. The host cells were lysed by treatment with Brucella broth containing 10% fetal bovine serum (FBS) for 30 min, and then all cells from the wells were scrubbed. The number of CFU for H. pylori was determined by serial dilution and spreading on appropriate selective medium as previously described after incubation for 4 to 7 days (23).

Statistical analysis
All of the experiments were performed in triplicate samples and error bars represent standard deviations. Differences with a P-value below 0.05 were considered statistically significant by paired T-Test. Normality of the data and the statistical analysis was performed using Minitab 17.

Effect of LAB and their CFS on the count of H. pylori
The count of H. pylori strains (Log 10 CFU/ml) in the presence of CFS of LAB and LAB are shown in Figure 1 (A-H). Results demonstrated that the reduction effect of CFS was higher than LAB. In the case of CFS was between 1-6 log but in the case of LAB was not more than 1 log. In the case of CFS of L. acidophilus ATCC 4356 and L. casei ATCC 39392 a reduction of 6-log was observed in H. pylori 23F but only a reduction of 1 log was observed in case H. pylori 23F in presence of L. ruteri ATCC 23272.

Effect of lactobacilli CFS on the urease activity of H. pylori
To perform this test, the optimized conditions  for urea concentration, pH of reaction buffer and temperature were obtained using RSM method. They were 15 %, 5.8 and 46-55 °C for urea concentration, pH, and temperature, respectively. The anti-urease activity effect of CFS was higher at concentrations of 30-40% than other concentrations, but the same effect was observed in these two concentrations. Figure

Effect of LAB on the adhesion of H. pylori to AGS cell line
By counting the number of adherent H. pylori to host cell AGS, the percentage of adherence was analyzed. We observed that adherence in the H. pylori HP1 and AS923 (20%) were inhibited by all of the LAB. However, L. rhamnosus ATCC 7469 had anti-adherent effect against five H. pylori strains, and L. acidophilus ATCC 4356 and L. reuteri ATCC 23272 had anti-adherent effect against four H. pylori strains. Adherence of H. pylori HP1 and AS923 was reduced in presence of all LAB but in the cases of H. pylori hp13 and 23F no decrease was found. Figure 3 showed CFU (log 10 /ml) of adherent bacteria to AGS in the presence and in absence of LAB.

Discussion
Treatment of H. pylori infection is often effectuated by routine triple therapy regimen and if the infecting strain is resistant, its successful eradication would be compromised (24). Nowadays, the rate of treatment failures is rising and the major cause for this would be bacterial resistance to frequently prescribed antibiotics,   so, it is important to know the pattern of this resistance. Furthermore, the antibiotic resistance patterns vary between countries and within different regions of the same country (25,26). The major pattern of antibioticresistance in H. pylori strains used in the present study was resistance to metronidazole, clarithromycin, amoxicillin, ampicillin, erythromycin, furazolidone, and azithromycin ( Table 1). Studies of researchers in Iran recommended fluoroquinolones for the treatment of H. pylori infection because of the inadequacy of the common antibiotics (27,28). This resistance pattern reflects the importance of antibiotic use in our country, especially in children (11). The results of a study on 218 isolates of H. pylori gathered from 985 dyspeptic patients during 2010-2017 in Iran, showed a considerable increase in resistance to presently used antibiotics such as metronidazole, ofloxacin, tetracycline, and clarithromycin (29). This high resistance to antibiotics, requires alternative ways to reduce the rates of H. pylori infection Probiotics bacteria are prescribed for the treatment of many gastrointestinal disorders, ranging from diarrhea to H. pylori infection (15). Researchers demonstrated an increase in the rate of H. pylori eradication due to the use of probiotics along with triple therapy (30). The invivo studies have shown that eradication rate of H. pylori infection by drugs can be improved by administration of lactobacilli and also due to reducing gastric mucosal inflammation (31)(32)(33)(34). The decrease of 6-log of H. pylori was reported in presence CFS of L. fermentum after 24 hr (35). Complete inhibition of H. pylori in vitro was observed in the case of co-incubation with probiotics (L. rhamnosus and L. acidophilus) at ratios of 1:10 and 1.100 (36). Takeda et al. reported that inhibitory activity of probiotic bacteria against H. pylori was straindependent so that L. paracasei strain 06TCa19 and L. plantarum strain 07MR044 exhibited potent abilities to inhibit the growth of H. pylori in co-culture method (22), that detected in the present study. Antagonistic effect of LAB related to resource competition, production different low molecular weight substances (e.g. diactyl, acetaldehyde, hydrogen peroxide, ect.); production of different organic acid (e.g. lactic, propionic, succinic and acetic); pH lowering effect and production of bacteeriocin and bacteriocin-like substances that are produced differently by LAB so the inhibitory effects of LAB on H. pylori differ from strain to strain (37,38).
In the present study, we used the LAB of human origin that has different metabolism pathway (fermentation pathways). Our results demonstrated that both CFS of LAB and LAB displayed anti-H. pylori effect but CFS had more effect than LAB. Both CFS and LAB caused a significant reduction in the growth of two H. pylori strains (23F and PR) which were highly resistant to antibiotics. Reducing the number of colonized bacteria may also be important in Quorum sensing (QS) process which is a regulatory mechanism used by bacteria to receive and respond to variations in cell-population density. This is effectuated through the expression of specific genes (39).
In the H. pylori genome, the only known QS gene is the luxS gene. LuxS has an alternative role in the regulation of motility (by modulating flagellar transcription and flagellar biosynthesis) and biofilm formation (40). The expression of this gene may be altered by decreasing the number of H. pylori which then can alter the regulation of motility and biofilm formation.
Higher anti-H. pylori effect of CFS observed may be due to the fact that CFS contains antimicrobial agents that causing death of H. pylori during the early growth time. The anti-H. pylori effect of CFS was lost or decreased when the pH of CFS adjusted to 7 (the data not showed). In a genotobiotic murine model, L. salivarius produces high levels of lactic acid and thereby inhibits growth of H. pylori, it has been suggested that the concentration of organic acids was related to anti-H. pylori activity of CFS of LAB (41,42). Inhibition of H. pylori by the production of lactic acid in L. salivarius, L. acidophilus, L. rhamnosus and L. casei strain Shirota has been reported (43), although was demonstrated that anti-inhibition effects of Lactobacillus strains were only partly explained by organic acid production (44). Coconier et al. showed that supernatant of L. acidophilus decreases the survival ability of H. pylori due to the presence of anti-Helicobacter substances produced by L. acidophilus that may be different from lactic acid (45). Different studies have used agar well diffusion assay to determine CFS susceptibility of H. pylori (16,37) but the results of our experiment, were not measurable due to the small diameters of inhibition zones.
Our results concerning CFS effect against urease activity of H. pylori strains showed that CFS of all LAB could reduce urease activity of H. pylori HP70, HP153, 157, 23F, AS923 and PR during 120 min. Urease activity is essential for initiating the stomach colonization of stomach by H. pylori which posses both cytoplasmic and surface-associated or extracellular urease and activity of surface-localized urease is essential for resistance of H. pylori to acid (46). In our study, CFS of probiotics bacteria could decrease urease activity but no inhibitory effect was found on urease activity when pH of CFS was neutralized with NaOH. Therefore, organic acids in CFS of LAB play an important role in the inhibition of urease activity of H. pylori strains. Also, inhibition effect of urease by acetic acid and lactic acid was observed in our work. The urease activity of H. pylori co-cultured with lactobacillus supernatants decreased (20). Lactic acid of L. femntum UCO-979C obtained from human gut could inhibit the urease enzyme of H. pylori strains (35). However, the bacteriocin of L. plantarum and L. acidophilus could inhibit or reduce the urease activity in Proteus spp (47).
Inhibitory effects of LAB on adherence of H. pylori strains to AGS cell line may reflect their in vivo effect that can help to prevent infection in an early stage of H. pylori colonization. The anti-adherence effects of probiotics can be produced by bacterial competition for binding sites on epithelial cells where at the same time the antimicrobial substances are also secreted by probiotic bacteria. L. reuteri possesses the cell surface proteins that inhibit H. pylori to bind to glycolipids receptor in vitro (48), also lactobacilli act directly on H. pylori by an effectors molecule that is released into the medium. This effectors molecule acts on H. pylori by inhibiting expression of the adhesion-encoding gene sabA (23). Animal studies demonstrated that prior colonization by H. pylori, probiotics can prevent H. pylori infection Effect of lactobacilli probiotics on H. pylori Rezaee et al. in germ-free mice (49). In our study, it seems that due to simultaneous exposure of H. pylori and LAB to AGS, competition between them to bind to the host cells may be produced, which is one of the inhibitory mechanisms.

Conclusion
Treatment of H. pylori using synthetic compounds is associated with several problems such as the high cost of medications, post-treatment bacterial resistance, and adverse side effects. Therefore, exploration of some safer and non-chemical anti-H. pylori compounds are becoming important as an alternative therapy against H. pylori infections. Probiotic bacteria can inhibit H. pylori by immunological and non-immunological mechanisms. Our study reviewed the non-immunological mechanisms that showed the anti-H. pylori effect of probiotics bacteria depends on the strain of H. pylori and LAB. As lactobacillus can reduce the growth of H. pylori, inhibit the activity of its urease, and reduce adhesion H. pylori to cell line, they can play an important role in preventing colonization of stomach by H. pylori.